Physiological significance of proline and glycinebetaine: Maintaining photosynthesis during NaCl stress in wheat

Experiments on the physiological significance of accumulation of proline and glycinebetaine (GB) in sustaining photosynthesis during salt stress in wheat in vivo showed that pre-treatment with GB, but not proline, alleviated NaCl-induced stomatal and non-stomatal inhibition of photosynthesis completely. A permeating and non-dissociating osmoticum, 3-orthomethyl-glucopyranose, also alleviated NaCl-induced perturbations of photosynthesis, suggesting that GB may work by maintaining chloroplast volume and not by specific effects on photosynthetic processes.     

Salicylic acid alleviates adverse effects of heat stress on photosynthesis through changes in proline production and ethylene formation

We investigated the potential of salicylic acid (SA) in alleviating the adverse effects of heat stress on photosynthesis in wheat (Triticum aestivum L.) cv WH 711. Activity of ribulose 1,5-bisphosphate carboxylase (Rubisco), photosynthetic-nitrogen use efficiency (NUE), and net photosynthesis decreased in plants subjected to heat stress (40°C for 6 h), but proline metabolism increased. SA treatment (0.5 mM) alleviated heat stress by increasing proline production through the increase in γ-glutamyl kinase (GK) and decrease in proline oxidase (PROX) activity, resulting in promotion of osmotic potential and water potential necessary for maintaining photosynthetic activity. Together with this, SA treatment restricted the ethylene formation in heat-stressed plants to optimal range by inhibiting activity of 1-aminocyclopropane carboxylic acid (ACC) synthase (ACS). This resulted in improved proline metabolism, N assimilation and photosynthesis. The results suggest that SA interacts with proline metabolism and ethylene formation to alleviate the adverse effects of heat stress on photosynthesis in wheat.     

Exogenous proline and glycinebetaine increase NaCl-induced ascorbate-glutathione cycle enzyme activities, and proline improves salt tolerance more than glycinebetaine in tobacco Bright Yellow-2 suspension-cultured cells

Up-regulation of the antioxidant system provides protection against NaCl-induced oxidative damage in plants. Antioxidants and activity of enzymes involved in the ascorbate-glutathione (ASC-GSH) cycle in tobacco Bright Yellow-2 (BY-2) were investigated to assess the antioxidant protection offered by exogenous proline and glycinebetaine (betaine from now on) against salt stress using cells grown in suspension culture. Reduced ascorbate (ASC) was detected in BY-2 cells but dehydroascorbate (DHA) was not. Large quantities of a reduced form of glutathione (GSH) and smaller quantities of an oxidized form of glutathione (GSSG) were detected in BY-2 cells. Salt stress significantly reduced the contents of ASC and GSH as well as activities of ASC-GSH cycle enzymes such as ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), and glutathione reductase (GR). Exogenous proline or betaine increased the activities of all enzymes except MDHAR involved in NaCl-induced ASC-GSH cycle. Levels of ASC and GSH in BY-2 cells under salt stress were lower in the presence of proline or betaine than in the absence of proline or betaine whereas there was no difference in redox status. Proline proved more effective than betaine in maintaining the activity of enzymes involved in NaCl-induced ASC-GSH cycle. Neither proline nor betaine had any direct protective effect on NaCl-induced enzyme activity involved in the antioxidant system; however, both improved salt tolerance by increasing enzyme activity. The present study, together with our earlier findings [Hoque MA, Okuma E, Banu MNA, Nakamura Y, Shimoishi Y, Murata Y. Exogenous proline mitigates the detrimental effects of salt stress more than exogenous betaine by increasing antioxidant enzyme activities. J Plant Physiol 2006;164:553-61.], suggests that proline offered greater protection against salt stress than betaine did because proline was more effective in increasing the activity of enzymes involved in the antioxidant system.     

Calcium chloride effects on salinity-induced oxidative stress, proline metabolism and indole alkaloid accumulation in Catharanthus roseus

Catharanthus roseus (L.) G. Don. plants were grown with NaCl and CaCl2 in order to study the effect of CaCl2 on NaCl-induced oxidative stress in terms of lipid peroxidation (TBARS content), H2O2 content, osmolyte concentration, proline (PRO)-metabolizing enzymes, antioxidant enzyme activities, and indole alkaloid accumulation. The plants were treated with solutions of 80 mM NaCl, 80 mM NaCl with 5 mM CaCl2 and 5 mM CaCl2 alone. Groundwater was used for irrigation of control plants. Plants were uprooted randomly on 90 days after sowing (DAS). NaCl-stressed plants showed increased TBARS, H2O2, glycine betaine (GB) and PRO contents, decreased proline oxidase (PROX) activity, and increased gamma-glutamyl kinase (gamma-GK) activity when compared to control. Addition of CaCl2 to NaCl-stressed plants lowered the PRO concentration by increasing the level of PROX and decreasing the gamma-GK activities. Calcium ions increased the GB contents. CaCl2 appears to confer greater osmoprotection by the additive role with NaCl in GB accumulation. The antioxidant enzymes superoxide dismutase (SOD), peroxidase (POX) and catalase (CAT) were increased under salinity and further enhanced due to CaCl2 treatment. The NaCl-with-CaCl2-treated C. roseus plants showed an increase in total indole alkaloid content in shoots and roots when compared to NaCl-treated and untreated plants.     

Proline and glycinebetaine enhance antioxidant defense and methylglyoxal detoxification systems and reduce NaCl-induced damage in cultured tobacco cells

Salt stress impairs reactive oxygen species (ROS) and methylglyoxal (MG) detoxification systems, and causes oxidative damage to plants. Up-regulation of the antioxidant and glyoxalase systems provides protection against NaCl-induced oxidative damage in plants. Thiol–disulfide contents, glutathione content and its associated enzyme activities involved in the antioxidant defense and glyoxalase systems, and protein carbonylation in tobacco Bright Yellow-2 cells grown in suspension culture were investigated to assess the protection offered by proline and glycinebetaine against salt stress. Salt stress increased protein carbonylation, contents of thiol, disulfide, reduced (GSH) and oxidized (GSSG) forms of glutathione, and the activity of glutathione-S-transferase and glyoxalase II enzymes, but decreased redox state of both thiol–disulfide and glutathione, and the activity of glutathione peroxidase and glyoxalase I enzymes involved in the ROS and MG detoxification systems. Exogenous application of proline or glycinebetaine resulted in a reduction of protein carbonylation, and in an increase in glutathione redox state and activity of glutathione peroxidase, glutathione-S-transferase and glyoxalase I under salt stress. Neither proline nor glycinebetaine, however, had any direct protective effect on NaCl-induced GSH-associated enzyme activities. The present study, therefore, suggests that both proline and glycinebetaine provide a protective action against NaCl-induced oxidative damage by reducing protein carbonylation, and enhancing antioxidant defense and MG detoxification systems.     

Role of nitric oxide under saline stress: implications on proline metabolism

The present work is focused on the possible relationship between nitric oxide and the induction of proline in response to salt stress. The plants were subjected to 100 mM NaCl and sodium nitroprusside (SNP; the donor of NO) at different concentrations. The plants showed lower NaCl-induced oxidative stress and proline accumulation after application of low concentrations of SNP together with the NaCl treatment. The reduction in the proline content was related to increased activity of proline dehydrogenase. These results suggest that the NO could be capable of mitigating damage associated with salt stress.     

根施甜菜碱对干旱胁迫下小麦幼苗类囊体膜组分和功能的影响

干旱胁迫造成两小麦品系类囊体膜上的单半乳糖脂甘油二酯(MGDG)、双半乳糖脂甘油二酯(DGDG)、磷脂酰胆碱(PG)以及叶绿素含量显著下降,甜菜碱预处理能缓解这些组分的下降.干旱胁迫下,抗旱型小麦品系HF9 70 3的硫代异鼠李糖甘油二酯(SQDG)、反式十六碳-烯酸[16:1(3t)]含量显著上升,MGDG中亚麻酸(18:3)相对含量显著下降;而干旱敏感型品系SN215953则表现为SQDG、16:1(3t)含量显著下降,MGDG中脂肪酸变化不明显,这可能是两个小麦品系抗旱性差异的重要原因之一;甜菜碱处理能显著减小干旱处理与对照之间的差异,且对SN215953的作用较HF9703大.另外,干旱胁迫引起类囊体膜上Ca2 -ATPase活性、Hill反应活性及叶片净光合速率下降,外源甜菜碱能缓解其下降趋势.     

Exogenously supplied compatible solutes rapidly ameliorate NaCl-induced potassium efflux from barley roots

It has been suggested that the role of compatible solutes in plant stress responses is not limited to conventional osmotic adjustment, but also includes some other regulatory or osmoprotective functions. In this study, we hypothesized that one such function is in maintaining cytosolic K+ homeostasis by preventing NaCl-induced K+ leakage from the cell, a feature that may confer salt tolerance in many species, particularly in barley. This hypothesis was investigated using the non-invasive microelectrode ion flux (MIFE) measuring technique. We show that low (0.5-5 mM) concentrations of exogenously supplied proline or betaine significantly reduced NaCl-induced K+ efflux from barley roots in a dose-response manner. This effect was instantaneous, implying that large intracellular concentrations of compatible solutes are not required for an amelioratory role. Exogenously supplied betaine also significantly enhanced NaCl-induced H+ efflux, but only in pre-incubated roots, implying some alternative mechanism of regulation. Sap K+ and Na+ analysis and membrane potential measurements are also consistent with the model that one function of compatible solutes is in maintaining cytosolic K+ homeostasis by preventing NaCl-induced K+ leakage from the cell, possibly through the enhanced activity of H+-ATPase, controlling voltage-dependent outward-rectifying K+ channels and creating the electrochemical gradient necessary for secondary ion transport processes. These data provide the first direct evidence for regulation of ion fluxes across the plasma membrane by physiologically relevant low concentrations of compatible solutes.     

Glycine betaine application modifies biochemical attributes of osmotic adjustment in drought stressed wheat

Nineteen wheat genotypes were used to examine the effects of foliar applied glycine betaine (GB, 100 mM) on concentration of various osmolytes (such as proline, choline, GB and sucrose) under drought stress conditions. Drought stress caused a significant increase in proline content and GB content of wheat genotypes, both at maximum tillering and anthesis stages. Choline and sucrose were accumulated significantly at higher levels under stress conditions at both the stages. GB application increased the proline content and endogenous levels of GB in comparison to their stressed counterparts both at maximum tillering and anthesis stages but this increase was observed to be genotype specific. Furthermore, significant decrease in choline levels and sucrose contents of GB treated plants at anthesis stage and enhanced levels of proline questioned about involvement of GB in production of other osmolytes as well as stage specific response of wheat genotypes to GB spray. But these changes in osmolyte accumulation (OA) were not correlated with relative water content and stress tolerance index observed, under both GB sprayed and non-sprayed drought stressed conditions. So OA could not be considered as a selection criteria for drought tolerance in wheat.     

Suppression of the osmoinduced proline response of rapeseed leaf discs by polyamines

Rapeseed leaf discs (RLD) subjected to upshock osmotic stress accumulate proline (Pro). Di‐ and polyamines (PA) supplied to the external medium suppressed Pro accumulation. These effects were dependent not only on diamine and PA concentrations but also on their cationic charge. The suppression of Pro accumulation required that diamine and PA be taken up and further accumulated in the leaf tissues. Glycine betaine (GB) also inhibited Pro accumulation, with the effects of GB and PA being additive. Experiments to elucidate the mechanism(s) responsible for the inhibitory effect of spermine (Spm) indicated that it could be simulated with methionine sulfoximine (MSO), a potent inhibitor of glutamine synthetase. The inhibitory effects of Spm and MSO were both alleviated by supplying glutamine to the RLD. In addition, Spm as well as MSO increased glutamate content, indicating that these compounds could inhibit the conversion of glutamate to proline. A comparison of the changes in chlorophyll and protein content of RLD osmotreated with or without added Spm indicates that this PA behaves as an antisenescent compound, preventing chlorophyll breakdown and proteolysis and hence the conversion of amino acids to Pro. Since the PA concentrations used in this work were much higher than the endogenous concentrations in RLD, the significance of PA under osmotic stress remains unclear. This study shows, however, that PA can suppress Pro accumulation.     

Tree photosynthesis modulates soil respiration on a diurnal time scale

To estimate how tree photosynthesis modulates soil respiration, we simultaneously and continuously measured soil respiration and canopy photosynthesis over an oak‐grass savanna during the summer, when the annual grass between trees was dead. Soil respiration measured under a tree crown reflected the sum of rhizosphere respiration and heterotrophic respiration; soil respiration measured in an open area represented heterotrophic respiration. Soil respiration was measured using solid‐state CO₂ sensors buried in soils and the flux‐gradient method. Canopy photosynthesis was obtained from overstory and understory flux measurements using the eddy covariance method. We found that the diurnal pattern of soil respiration in the open was driven by soil temperature, while soil respiration under the tree was decoupled with soil temperature. Although soil moisture controlled the seasonal pattern of soil respiration, it did not influence the diurnal pattern of soil respiration. Soil respiration under the tree controlled by the root component was strongly correlated with tree photosynthesis, but with a time lag of 7–12 h. These results indicate that photosynthesis drives soil respiration in addition to soil temperature and moisture.     

Exogenous Glycine Betaine Reduces Drought Damage by Mediating Osmotic Adjustment and Enhancing Antioxidant Defense in <i>Phoebe hunanensis</i>

Drought stress negatively impacts growth and physiological processes in plants. The foliar application of glycine betaine (GB) is an effective and low-cost approach to improve the drought tolerance of trees. This study examined the effect of exogenously applied GB on the cell membrane permeability, osmotic adjustment, and antioxidant enzyme activities of Phoebe hunanensis Hand.-Mazz under drought stress. Two levels (0 and 800 mL) of water irrigation were tested under different applied GB concentrations (0, 50, 100, and 200 mM). Drought stress decreased the relative water content by 58.5% while increased the electric conductivity, malondialdehyde, proline, soluble proteins, soluble sugars, and antioxidant enzyme activities (superoxide dismutase, catalase, peroxidase) by up to 62.9%, 42.4%, 87.0%, 19.1%, 60.5%, 68.3%, 71.7%, and 83.8%, respectively, on the 25^th day. The foliar application of GB, especially at 100 mM, increased the relative water content of P. hunanensis leaves under drought stress. The concentration of GB from 50 to 100 mM effectively alleviated the improvement of cell membrane permeability and inhibited the accumulation of membrane lipid peroxidation products. Under drought stress, the concentrations of proline, soluble proteins, and soluble sugars in the leaves of P. hunanensis increased as the applied GB concentration was increased and the water stress time was prolonged. Exogenously applied GB decreased oxidative stress and improved antioxidant enzyme activities as compared with treatments without GB application. Furthermore, the physiological and biochemical indexes of P. hunanensis showed a certain dose effect on exogenous GB concentration. These results suggest that GB helps maintain the drought tolerance of P. hunanensis.     

Proline suppresses Rubisco activity by dissociating small subunits from holoenzyme

Proline caused irreversible inhibition (involving reduction in V(max) without altering K(m) for RuBP) in Rubisco activity. Proline-induced suppression in Rubisco activity did not exceed beyond approximately 65% of the original activity even upon exposure to higher levels of proline for prolonged duration. However, NaCl-induced reduction in Rubisco activity was reversible. Native PAGE analysis of Rubisco-incubated with proline showed the presence of two distinct bands corresponding to approximately 430 and approximately 28 kDa, but that incubated with NaCl showed a single band. SDS-PAGE analysis revealed that the approximately 430- and approximately 28-kDa bands represent octamers of large subunits and dimers of small subunits, respectively. These results demonstrated for the first time that proline suppresses Rubisco activity by bringing about dissociation of the small subunits from the octamer core of large subunits, probably by weakening hydrophobic interactions between them.     

水分胁迫对冬小麦叶片CO2／H2O交换参数的影响

Changes of CO2/H2O exchange parameters were continually measuredin winter wheat under different water stress stages.The results showed that photosynthesis rate and transpiration rate of winter wheat in water stress conditions were obviously lower than that in non-stress conditions.After water stress,both of them slowly increased and even overtook that on sufficient irrigation treatment. Responses of winter wheat to water stress in different growth stages were different.To some extent, water stress can improve crop water use efficiency,speed up the process of milking.Under water stress condition,stomatal conductance limited diurnal changes of photosynthesis and transpiration in the morning but not in the afternoon.Transpiration is more sensitive to water stress than photosynthesis.     

Temperature response of in vivo Rubisco kinetics and mesophyll conductance in Arabidopsis thaliana: comparisons to Nicotiana tabacum

Biochemical models are used to predict and understand the response of photosynthesis to rising temperatures and CO₂ partial pressures. These models require the temperature dependency of ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco) kinetics and mesophyll conductance to CO₂ (g_m). However, it is not known how the temperature response of Rubisco kinetics differs between species, and comprehensive in vivo Rubisco kinetics that include g_m have only been determined in the warm‐adapted Nicotiana tabacum. Here, we measured the temperature response of Rubisco kinetics and g_m in N. tabacum and the cold‐adapted Arabidopsis thaliana using gas exchange and ¹³CO₂ isotopic discrimination on plants with genetically reduced levels of Rubisco. While the individual Rubisco kinetic parameters in N. tabacum and A. thaliana were similar across temperatures, they collectively resulted in significantly different modelled rates of photosynthesis. Additionally, g_m increased with temperature in N. tabacum but not in A. thaliana. These findings highlight the importance of considering species‐dependent differences in Rubisco kinetics and g_m when modelling the temperature response of photosynthesis.     

水分胁迫对冬小麦叶片CO_2/H_2O交换参数的影响

水资源严重匮乏已成为华北平原农业可持续发展的主要障碍因素 [1] ,提高有限水资源的利用效率显得十分重要。以前的研究主要注重农田水平作物与水分的关系 [2 ,4 ] ,利用作物生物学进行节水研究不够 [3,4 ] 。Roa等人认为作物适度的水分亏缺可获得高产 [15] ;Jensen等人认为适度水分胁迫甚至能使作物水分利用效率显著提高 [5,6] ,依此发展了调亏灌溉思想 ,对有限水量在作物生育期内时空最优分配制度进行研究 ,目前已为世界各国广泛关注 [6] 。作物 CO2 /H2 O交换参数包括光合速率、蒸腾速率、水分利用效率等 ,这些是确定作物水分高效利用…     

Photosynthesis of <Emphasis Type="Italic">Rehmannia glutinosa</Emphasis> subjected to drought stress is enhanced by choline chloride through alleviating lipid peroxidation and increasing proline accumulation

Rehmannia glutinosa seedlings were pretreated with choline chloride (CC) in concentrations of 0, 0.7, 2.1 and 3.5 mM, and then subjected to drought and rewatering treatment to study the effects of CC on the generation of reactive oxygen species (O₂⁻, H₂O₂), lipid peroxidation, proline accumulation, water status and photosynthesis. The results showed that pretreatment with CC alleviated the inhibition of SOD and APX activity caused by drought stress, and therefore, the rate of O₂⁻ production and H₂O₂ concentration were reduced and lipid peroxidation decreased in pretreated plants. CC pretreatment also accelerated accumulation of proline, maintained higher Ψw and RWC, deferred leaf water loss during drought stress and retarded the drop in proline concentration after rewatering. Consequently, drought-induced decreases in F_m/F₀, F_v/F_m, Φ_PS2, q_P, and A and increase in q_NP were inhibited and the recovery of photosynthesis after rewatering was quicker in pretreated plants. Although differences in F_v/F_m, Φ_PS2 and q_P between treatments were not significant, there was a general trend that the effects of CC increased with the rise of its concentrations. The data suggested that 2.1 mM of CC be suitable for alleviating lipid peroxidation, promoting proline accumulation, retarding leaf water loss and improving photosynthesis of R. glutinosa seedlings under drought stress.     

Comparative physiological effects of three allelochemicals and two herbicides on <Emphasis Type="Italic">Dactylis glomerata</Emphasis>

Effects of three allelochemicals (2-benzoxazolinone (BOA), p-hydroxybenzoic, and ferulic acid) and two herbicides (linuron and fluometuron) on chlorophyll fluorescence, photosynthesis, accumulation of free proline and polyamines, and total content of soluble proteins were measured to assess herbicidal activity on Dactylis glomerata. The application of chemicals generally reduced Fv/Fm (more than 25 % with p-hydroxybenzoic acid, BOA, or linuron). BOA decreased the quantum efficiency of PSII temporarily, but it did not affect photosynthesis. Polyamine content was very low, while proline content was markedly affected by some of the chemicals, thus producing some osmotic adjustment. Ferulic acid did not cause any physiological effect at all. BOA and p-hydroxybenzoic acid caused significant changes in various physiological traits of Dactylis glomerata, specially interfering with the quantum efficiency of the PSII.     

Roles of glycine betaine and proline in improving plant abiotic stress resistance

Glycine betaine (GB) and proline are two major organic osmolytes that accumulate in a variety of plant species in response to environmental stresses such as drought, salinity, extreme temperatures, UV radiation and heavy metals. Although their actual roles in plant osmotolerance remain controversial, both compounds are thought to have positive effects on enzyme and membrane integrity along with adaptive roles in mediating osmotic adjustment in plants grown under stress conditions. While many studies have indicated a positive relationship between accumulation of GB and proline and plant stress tolerance, some have argued that the increase in their concentrations under stress is a product of, and not an adaptive response to stress. In this article, we review and discuss the evidence supporting each of these arguments. As not all plant species are capable of natural production or accumulation of these compounds in response to stress, extensive research has been conducted examining various approaches to introduce them into plants. Genetically-engineered plants containing transgenes for production of GB or proline have thus far faced with the limitation of being unable to produce sufficient amounts of these compounds to ameliorate stress effects. An alternative “shot-gun” approach of exogenous application of GB or proline to plants under stress conditions, however, has gained some attention. A review of the literature indicates that in many, but not all, plant species such applications lead to significant increases in growth and final crop yield under environmental stresses. In this review article, numerous examples of successful application of these compounds to improve plant stress tolerance are presented. However, to streamline useful and economic applications of these compounds, further investigations are needed to determine the most effective concentrations and number of applications as well as the most responsive growth stage(s) of the plant. All these factors may vary from species to species. Furthermore, a better understanding of the mechanisms of action of exogenously applied GB and proline is expected to aid their effective utilization in crop production in stress environments.     

The influence of elevated CO2 on community structure, biomass and carbon balance of mediterranean old-fleld microcosms

We studied the effects of a doubling of atmospheric CO₂ concentration on intact monoliths of Mediterranean grassland in growth chambers where climatic field conditions were simulated. During the six month growing season, changes in community structure were monitored by quantifying species richness and cover. The CO₂ exchange of microcosms was measured continuously and the resulting quantity and quality of biomass were evaluated. Species richness and cover did not respond to elevated CO₂. After one month of treatment, CO₂ exchange measured during the day did not differ between CO₂ levels but the night respiration was two-fold higher under elevated CO₂. Stimulations of both day and night CO₂ flux by short-term CO₂ enrichment were recorded several times during the growing season. These results suggest that despite some downward adjustment of photosynthesis, net canopy photosynthesis was stimulated by elevated CO₂, but this stimulation was compensated for by an increased respiration. The 20% stimulation of final phytomass under elevated CO₂ was not significant: it resulted from unchanged live plant matter but a significant, 100% increase in litter accumulation. These results suggest that in low-productivity Mediterranean herbaceous systems, the greatest effect of CO₂ is not on the storage of carbon in biomass but on the turnover of the carbon in the plants.